The present invention relates to the dispensing of substances, such as biological reagents and samples. More particularly, the invention provides an apparatus and method for transferring small volumes of substances onto one or more substrates.
As the sensitivity of analytical techniques continues to improve, it is increasingly desirable to carry out chemical and biochemical assays using very small volumes of samples/reagents. This is especially true in situations involving expensive substances. Accordingly, it is now popular to utilize very small volumes of such substances laid down as xe2x80x9cspotsxe2x80x9d on the surface of a substrate, such as a slide, micro-card, chip or membrane.
Not only is it often desirable to provide ultra-small volumes of individual samples and/or reagents in the form of spots, it is becoming increasingly popular to arrange numerous such spots in close proximity to one another in the form of an array on a substrate. For example, a lab technician might need to evaluate a specimen for the presence of a wide assortment of target biological and/or chemical compounds, or to determine the reaction of many different specimens against one or more reagents, such as labeled probes. High-density array formats, or xe2x80x9cmicroarrays,xe2x80x9d permit many reactions to be carried out in a highly parallel fashion, saving space, time and money.
A variety of methods are currently available for making microarrays. Microarrays can be made, for example, by a robotic arm device having a spotting tip that moves successively between a sample-pickup well in a sample array, e.g., a microtitre plate, and a selected array position. Although high-density arrays of selected substances can be constructed by this approach, the production time and efficiency is limited by the fact that the regions of the microarray (or microarrays, if several are being constructed at once) are deposited one-by-one in a serial fashion. Additional time and effort is required where a plurality of different substances are laid down in the array, as the spotting tip must be cleaned and dried prior to being used with each new substance.
Multi-channel micropipette devices are available for laying down several reagent spots at once. Devices of this type typically have 8 or 12 micropipettes, fixed side-by-side in a linear array. Generally, these devices are unsuitable for quickly producing very dense arrays, as the size of each micropipette and any associated service connections (e.g., supply tubing, electrical connections, etc.) limits the minimal center-to-center spacing (pitch) that can be achieved for adjacent spots. Also, since only a few spots (usually 8 or 12) can be laid down at a time with such devices, the production of very dense arrays, e.g., having hundreds or thousands of spots with a submillimeter pitch, tends to be a very tedious and time-consuming process.
Another technique employs an array of pins arranged to simultaneously dip into an array of reservoirs, e.g., the 96 wells of a microtitre plate, to pick up one or more selected substances for transfer to a substrate, such as a membrane. Similar to the multi-channel pipette devices, the pitch spacing is limited by the size of each pin. Also, the pins of such arrays are typically arranged to match the pitch of a conventional supply-well array, typically 2xc2xc, 4xc2xd, or 9 mm center-to-center. Thus, similar to the multi-channel pipetters, the production of very dense arrays can only be accomplished by sequentially laying down a number of sub-arrays, e.g., in a staggered or interleaved fashionxe2x80x94a very cumbersome and inefficient endeavor.
As an additional disadvantage, most of the known spotting techniques require the handling or transfer of substances between multiple receptacles (e.g., pipettes, flasks, vials, etc.) and/or flow lines (e.g., channels, hoses, tubing). Such transfers frequently result in a loss or contamination of the substance, thereby reducing the overall efficiency and sensitivity of the assay. Particularly with regard to expensive substances, it is generally desirable to keep such losses to a minimum.
In view of the above, the need is apparent for a device and method useful for delivering a micro-volume of a substance onto a substrate in a quick and efficient manner. Preferably, the device should be relatively easy to use, cost effective and readily adaptable for the production of micro-arrays having a great number of individual spots.
In one of its aspects, the present invention provides an apparatus for spotting a selected substance (e.g., a liquid sample or reagent, or micro-particles such as beads) onto one or more substrates.
In one general embodiment, the apparatus of the invention includes a base, adapted to hold one or more reagents, and a conveyor. The conveyor includes a movable surface defining (i) a plurality of spaced, tandemly-arranged substrate-support regions, each of which is adapted to support a substrate, and (ii) an opening between each adjacent pair of substrate-support regions. The conveyor is operable to advance the substrate-support regions along a transport pathway extending over the base. Further included is a transfer instrument or head having a spotting tip mounted for movement along an axis, toward and away from a raised position at which the tip is disposed above the conveyor surface. Shifting means, e.g., an actuator (such as a solenoid, or the like), are operatively connected to the tip for moving the same along its axis. A control unit is operatively connected to the conveyor and the actuator. At the direction of the control unit, a selected opening of the conveyor surface can be advanced to a position generally aligned with the axis of the transfer tip, at which point the control unit can signal the shifting means to shift the tip away from its raised position through such opening to contact reagent in the base. The shifting means can then withdraw the tip from the reagent and through the opening by shifting the tip toward its raised position. A selected site of a substrate-support region upstream of the selected opening can then be advanced to a position generally aligned with the axis of the transfer tip, at which point the control unit can signal the shifting means to shift the tip away from its raised position toward such site to transfer a selected amount of reagent from the tip to a selected region of a substrate at the substrate-support region.
According to one embodiment, one or more additional transfer heads and associated shifting means are disposed at spaced positions along the transport pathway, and structure is provided in the base for holding one or more reagents at each of the spaced positions. Such structure can include, for example, one or more tube holders (e.g., apertures or bores formed along a top surface of the base). The various transfer heads can be positioned along a line running parallel with the transport pathway, or one or more of the transfer heads can be laterally offset from the other transfer heads.
In one embodiment, a plurality of transfer heads are disposed in a row extending laterally or obliquely across the conveyor surface at one or more of the spaced positions along the transport pathway.
One embodiment contemplates a channel or cavity extending through at least a portion of the base. For example, an elongate channel can extend longitudinally through a central region the base. Optionally, a flow line can communicate a remote fluid source with the channel. In one such arrangement, a fluid flow line is connected to a fitting at one end of the channel. An outlet of the flow line is arranged so as to direct a selected fluid, passed through the line, into and along the channel. The channel can further include an egression port, e.g., at a distal end, through which any fluid(s) directed into the channel can exit.
In one embodiment, the base of the apparatus is adapted to hold one or more reagent reservoirs (e.g., tubes, vials, or the like) such that a lower region of each reservoir extends at least partially into a channel of the base, such as the channel just described. For example, apertures can be formed along the top of the base into which respective reagent-holding tubes can be inserted. Each aperture, in this exemplary construction, communicates the interior of the channel with a region between the base and the transport pathway. With the tubes in place along the base, a cooling fluid (e.g., a gas, or water) passed through the channel can impinge upon the accessible external surfaces of the tubes, thereby cooling the tubes so as to discourage evaporation of the reagent(s) held therein.
According to one exemplary design, the transfer tip, when shifted away from its raised position, with its axis of motion unobstructed (i.e., through an opening defined by the conveyor surface), is adapted to enter at least partially into a channel extending through the base. At this position, a cleaning fluid (e.g., a liquid solvent) passed through the channel can clean the tip. Optionally, a dry, warm gas subsequently passed through the channel can be used to dry the cleaned tip.
Any suitable transfer instrument or head can be used, including contact and/or non-contact type devices. For example, the apparatus can employ a transfer head having an elongated tip in the nature of a pin or rod. In a typical construction, a relatively narrow rod is employed, e.g., one having a distal end less than about 500 xcexcm in diameter, and preferably less than about 250 xcexcm in diameter. In another exemplary arrangement the tip includes a channel of capillary size (e.g., less than about 1 mm in diameter) adapted to draw in a liquid reagent, when shifted into contact therewith, by way of capillary action. Still further embodiments contemplate the use of a micropipette, syringe device, jetting apparatus, or other xe2x80x9csip and spitxe2x80x9d assembly, as the transfer tip.
Preferably, the transfer tips of the transfer head or instrument are of an independent construction. The tips are not permanently fixed spatially with respect to one another. Each individual transfer tip can be attached to and detached from the head, without affecting or otherwise disturbing any other transfer tip(s) of the apparatus.
One embodiment of the invention teaches a transfer head having a plurality of spotting tips mounted side-by-side, in spaced relation. Each tip, in this embodiment, is adapted for movement along a respective axis, toward and away from a raised position at which the tip is disposed above the conveyor surface.
The conveyor of the transfer apparatus can be, for example, a linear-type conveyor or a carousel-type arrangement, among others. In one embodiment, the conveyor surface takes the form of an elongate web. Each of the substrate-support portions of the web, in this embodiment, defines a substrate portion or region that can be spotted. That is, the web and substrates are of an integral construction. In one particular arrangement, the web material is a flexible, membrane material. In another embodiment, the conveyor surface takes the form of an elongate flexible belt, e.g., a rubber or metallic endless belt, upon which separately formed substrates (e.g., 1xe2x80x3xc3x973xe2x80x3 micro-cards) can be removably placed. In one particular arrangement, the belt includes a pocket at each of its substrate-support regions for receiving respective micro-cards and maintaining the position of each at a known location as it is advanced along the transport pathway and spotted.
Another general embodiment of the spotting apparatus, as taught herein, includes a conveyor belt comprising a plurality of substrate-support regions separated from one another by intervening open regions therebetween. A base is located beneath the conveyor belt for supporting one or more reagent reservoirs (e.g., tubes, vials, or the like). A transfer instrument or head is disposed above the base and the conveyor belt, having a spotting tip mounted for movement between (1) a raised position above the conveyor belt, (2) a reagent dispensing position for depositing reagent on a substrate carried by one of the substrate-support regions, and (3) an extended position below the conveyor belt which is achieved by passing the tip through one of the open regions. Further included are means for moving the conveyor belt (including, for example, a motor, drive train, and driven roller) along a transport pathway such that the substrate-support regions pass generally along a plane extending between the base and the transfer head. Shifting means (e.g., an actuator, such as a z-motion actuator) are operatively connected to the spotting tip for shifting it between the extended, reagent dispensing, and raised positions. One or more controllers are operatively connected to the moving means and shifting means, the controllers being operable to (i) shift the spotting tip from its raised position to its extended position by traversing a selected open region in the conveyor belt, for withdrawing reagent from a reservoir supported by the base, (ii) raise the spotting tip after step (i) to a position above the conveyor belt, (iii) move the conveyor belt so that a selected substrate is positioned below the raised spotting tip, (iv) move the spotting tip to a reagent dispensing position so that reagent is deposited onto a selected region of the selected substrate, (v) after reagent deposition, raise the spotting tip to its raised position, (vi) move the conveyor belt so that the spotting tip is positioned above another open region. If desired, the controller can repeat steps (i)-(vi) a selected number of times.
A further general embodiment of a spotting apparatus, as taught herein, includes a base, adapted to hold a reagent, and a conveyor. The conveyor includes a surface defining (i) a plurality of spaced, tandemly-arranged substrate regions, and (ii) an opening between adjacent substrate regions. The conveyor is operable to advance such regions along a transport pathway extending over the base. A transfer instrument or head is provided, having a spotting tip mounted for movement along an axis, toward and away from a raised position at which the tip is disposed above the conveyor surface. Shifting means, e.g., an actuator, are operatively connected to the tip for moving the same along its axis. A control unit is operatively connected to the conveyor and the shifting means. At the direction of the control unit, a selected opening of the conveyor surface can be advanced to a position generally aligned with the axis of the transfer tip, at which point the control unit can signal the shifting means to shift the tip away from its raised position through such opening to contact reagent in the base. The shifting means can then withdraw the tip from the reagent and through the opening by shifting the tip toward its raised position. A selected site of a substrate region, upstream of the selected opening, can then be advanced to a position generally aligned with the axis of the transfer tip, at which point the control unit can signal the shifting means to shift the tip away from its raised position toward such site to transfer a selected amount of reagent from the tip thereto.
In one particular arrangement of the spotting apparatus, the conveyor surface is a flexible web material, such as a membrane or the like.
In another of its aspects, the present invention provides a method for spotting a selected substance (or substances) onto one or more substrates.
According to one general embodiment, the method includes the steps of:
(i) advancing a plurality of spaced, tandemly-arranged substrates along a transport pathway extending over a reagent-supply location;
(ii) from a position over the reagent-supply location and the pathway,
(a) extending a reagent-transfer instrument, or tip, through an intervening region separating an adjacent pair of advancing substrates to contact reagent held at the reagent-supply location,
(b) withdrawing the reagent-transfer instrument, along with a portion of such reagent, through the intervening region to a position above the transport pathway, and
(c) transferring a selected amount of reagent from the reagent-transfer instrument onto a selected region of a selected substrate upstream of the intervening region.
In one embodiment, the substrates are integrally formed as spaced-apart expansive portions provided along an elongate web of material (e.g., a membrane material), and each of the intervening regions is an opening formed through the web of material (e.g., a cut-out region) between adjacent substrate portions.
In another embodiment, the substrates are advanced using a conveyor having a belt (e.g., a flexible endless belt) with a plurality of tandemly-arranged substrate-support regions. Each of the substrates, in this embodiment, is placed at a respective one of the substrate-support regions.
According to one embodiment, the transport pathway extends over a plurality of reagent-supply locations, disposed at spaced positions along the pathway. Step (ii), in this embodiment, is performed at two or more of the spaced positions in a fashion effective to produce a plurality of reagent spots on the selected substrate. The reagent spots can be placed along a line extending substantially parallel to the transport pathway, and/or one or more of the reagent spots can be placed at positions that are laterally offset from the other reagent spots.
In one embodiment, step (ii) is performed at least twice, in a substantially parallel fashion, using separate reagent-transfer instruments at one or more of the spaced positions.
Another embodiment contemplates the additional steps of: removing any reagent(s) being held at the reagent-supply location(s); extending at least a portion of each reagent-transfer instrument into a respective reagent-supply location; and flowing a cleaning fluid (e.g., a liquid solvent) along the reagent-supply location so that it contacts and cleans each reagent-transfer instrument or tip. Optionally, a drying fluid (e.g., a warm, dry gas) can be passed along the reagent-supply location, subsequent to such cleaning step, such that it contacts and dries each transfer instrument.
In one embodiment, one or more reagent reservoirs, or vessels, are placed at respective reagent-supply locations; and a cooling fluid is passed along the reagent-supply location so that it contacts the vessel, thereby reducing evaporative loss of any liquid reagent held therein.
A further embodiment contemplates, prior to step (i), the additional step of retrieving a vessel containing a selected reagent from a storage location, and placing the vessel at a reagent-supply location; and, subsequent to step (ii), the step of retrieving the vessel from the reagent-supply location, and returning the vessel to its storage location. In this way, the use of intermediate vessels, and consequent loss of reagent, is avoided.
Still a further aspect of the invention provides a substrate, bearing one or more reagent spots (e.g., a micro-array), produced in accordance with the method taught herein.
These and other features and advantages of the present invention will become clear from the following description.